Process for the manufacture of probiotic cheese

ABSTRACT

A process for the manufacture of a probiotic cheese, such as Cheddar cheese, comprises adding a 0.0-5.5% inoculum of a strain of  Lactobacillus paracasei , which is non-pathogenic, acid and bile tolerant and adherent to human epithelial cells, as a starter adjunct to cheese milk, said  L. paracasei  strain being capable of growing during the ripening phase to a level of 10 7  cfu/g or greater. The  L. paracasei  strains are found to grow and proliferate to high cell numbers (in excess of 10 8  cfu/g) in the cheese over eight months of ripening, even when added at a relatively low inoculum. The presence of the  L. paracasei  strains is found to have negligible effects on cheese composition, flavor and aroma.

This application is the national phase under 35 U.S.C. §371 of PCTInternational Application No. PCT/IE99/00047 which has an Internationalfiling date of May 26, 1999, which designated the United States ofAmerica.

TECHNICAL FIELD

This invention relates to the manufacture of probiotic cheese and, inparticular, to the manufacture of a probiotic cheese which contains atthe time of consumption a viable, actively growing strain of an addedbacterium.

BACKGROUND ART

The importance of probiotic-containing products to maintenance of healthand well-being is becoming a key factor affecting consumer choice,resulting in rapid growth and expansion of the market for such products,in addition to increased commercial interest in exploiting theirproposed health attributes. The majority of probiotic foods already onthe market, such as fermented milks and yoghurt are fresh products andare generally consumed within days or weeks of manufacture. In contrast,hard cheeses, such as Cheddar have long ripening times of up to twoyears.

Probiotic bacteria are described as ‘living’ micro-organisms, which uponingestion in certain numbers exert health benefits beyond inherent basicnutrition. Probiotics may be consumed either as a food component or as anon-food preparation. Foods containing such bacteria fall within the‘functional foods’ category and these are described as ‘foods claimed tohave a positive effect on health’. Such products are gaining morewidespread popularity and acceptance throughout the developed world andare already well accepted in Japan and the USA. Furthermore, increasedcommercial interest in exploiting the proposed health attributes ofprobiotics has contributed in a significant way to the rapid growth andexpansion of this sector of the market.

The potential health-promoting effects of dairy products whichincorporate probiotic organisms such as Lactobacillus andBifidobacterium spp. has stimulated a major research effort in recentyears. To date, the most popular food delivery systems for thesecultures have been freshly fermented dairy foods, such as yoghurts andfermented milks, as well as unfermented milks with cultures added.

There are relatively few reports concerning cheese as a carrier ofprobiotic organisms, even though there are a small number of ‘probioticcheeses’ currently on the market.

In 1994, Dinakar and Mistry (J. Dairy Sci. 77:2854-2864) incorporatedBifidobacterium bifidum into Cheddar cheese as a starter adjunct. Thisstrain survived well in the cheese and retained a viability ofapproximately 2×10⁷ cfu/g even after 6 months of ripening, withoutadversely affecting cheese flavour, texture or appearance. This examplesuggested that Cheddar could provide a suitable environment for themaintenance of probiotic organisms at high levels over long timeperiods. However, no growth of the B. bifidum was observed in the cheeseduring the ripening period and thus it is important to emphasise thatthe Bifidobacterium strain did not grow during manufacture and/orripening and thus had to be added at a relatively high inoculum. Inanother study, bifidobacteria were used in combination with Lb.acidophilus strain Ki as a starter in Gouda cheese manufacture (Gomes,A. M. P. et al. (1995); Neth. Milk Dairy J. 49:71-95). The two strainswere used as sole starters, requiring relatively large inocula (3%) ofboth strains and adaptation of cheese making technology. In this case,there was a significant effect on cheese flavour in the resultantproduct after 9 weeks of ripening, possibly due to acetic acidproduction by the bifidobacteria.

In order to exert a probiotic effect, cultures must maintain theirviability in food products through to the time of consumption, which forCheddar cheese is many months after manufacture.

Cheese is a milk product in which the whey protein/casein ratio does notexceed that of milk and which is obtained by coagulation of milk by theaction of rennet, followed by whey drainage. Starter cultures containinglactic acid bacteria are initially required during cheese making tometabolise lactose, thereby producing lactic acid and reducing the pH.During Cheddar cheese manufacture for example, the starter lactococcigrow, reaching maximum levels of approximately 10⁹ to 10¹⁰ cfu/g atsalting. Conditions in the cheese however, such as high salt in moisture(S/M), low pH, lack of a fermentable carbohydrate and low temperature ofripening can result in a dramatic decline in starter numbers during theearly weeks of ripening. The rate of decline depends on a number ofcharacteristics of the strain, including autolytic properties, salttolerance and phage resistance. In the meantime, a population ofnon-pathogenic organisms, referred to as non-starter-lactic acidbacteria (NSLAB), chiefly composed of lactobacilli (Lb. plantarum, caseiand brevis) and pediococci (Pediococcus pentosaceus) proliferate as thecheese ripens, a process that is generally performed at 2-16° C. It isbelieved that NSLAB gain access tothe cheesemilk during themanufacturing stage or that they survive pasteurisation in an attenuatedstate. Regardless, their numbers increase rapidly reaching maximumlevels of 10⁷ to 10⁸ cfu/g in ripened Cheddar cheese. Indeed, in maturecheese, NSLAB may represent the principal flora. Their role indetermining cheese quality remains unclear. NSLAB are generallyenumerated using an aerobic plate count on Rogosa or LactobacillusSelective (LBS) agar.

It may not be cost-effective to add probiotic strains to cheese inamounts corresponding to that finally required for a probiotic productat time of consumption. Rather what is required is a probiotic strainwhich can be added as a starter adjunct at a low inoculum to cheese andwhich grows to the required values of ˜>10⁷ cfu/g.

What is required, therefore, for a probiotic cheese with a long ripeningtime such as Cheddar is a probiotic strain which can survive and growthroughout manufacture and the ripening period.

DISCLOSURE OF INVENTION

The invention provides a process for the manufacture of a probioticcheese, which process comprises adding a 0.05-0.5% inoculum of a strainof Lactobacillus paracasei isolated from the human gastrointestinaltract, which is non-pathogenic, acid and bile tolerant and adherent tohuman epithelial cells, as a starter adjunct to cheese milk, said L.paracasei strain being capable of growing during the ripening phase to alevel of 10⁷ cfu/g or greater.

We have found that said strain of L. paracasei has the ability tosurvive the cheese manufacturing process and the capacity to grow andsurvive during the ripening/storage period. The strain of L. paracaseiused in the process according to the invention also has the ability tosurvive passage through the gastrointestinal tract as hereinafterdemonstrated. The presence of the added L. paracasei strain has beenfound to have negligible effects on cheese composition, flavour andaroma.

Preferably, a 0.1-0.25% inoculum of the L. paracasei is added to thecheese milk.

Also, preferably the ripening phase is at least six months.

Further, preferably, the ripening phase is eight months or greater.

The L. paracasei strains used in the process according to the inventionhave been found to grow and proliferate to high cell numbers in cheeseover eight months of ripening, when added at a low inoculum as describedherein.

Thus, in one embodiment of the invention, the L. paracasei is capable ofgrowing during the ripening phase to a level of 10⁸ cfu/g or greater.

Preferably, the L. paracasei is tolerant to temperatures of 37° C. orgreater.

Also, preferably the L. paracasei can be enumerated and distinguishedfrom the resident flora.

Most preferably, the added L. paracasei cells are enumerated anddistinguished by a randomly amplified polymorphic DNA (RAPD) methodwhich allows the generation of discrete DNA fingerprints for therespective strains.

The RAPD used allowed the generation of discrete DNA fingerprints foreach strain which were clearly distinguishable from those generated bythe natural flora of the cheeses.

Preferably, the cheese manufactured is a hard cheese.

In an especially preferred embodiment the cheese is Cheddar cheese.

Cheddar cheese has particular advantages as a carrier of a probioticmicro-organism as described herein. Having a higher pH than the moretraditional probiotic foods (e.g. yoghurts and fermented milks), itprovides a more stable milieu to support their long-term survival.Furthermore, the matrix of the cheese and its relatively high fatcontent offers protection to probiotic bacteria during passage throughthe gastrointestinal tract (GIT).

The L. paracasei strains used in accordance with the invention wereobtained from University College Cork, under a restricted MaterialsTransfer Agreement, together with a number of other strains for thepurposes of investigation as described in the Examples.

The L. paracasei strains were found to have the requisite properties foruse in cheese manufacture whereas, for example, the Lactobacillussalivarius strains investigated died during the ripening period.

The L. paracasei strains used herein have the requisite ability toinfluence the microflora of both the cheese and the GIT, the ability ofthe culture to grow in dairy-based media, such as whey and phageinhibitory media and the ability of the culture to survive and/or growduring manufacture and throughout the shelf-life of the cheese product.

The invention also provides Lactobacillus paracasei strain NFBC 338.

Also the invention provides Lactobacillus paracasei strain NFBC 364.

Samples of these bacteria have been deposited at The NationalCollections of Industrial and Marine Bacteria Limited (NCIMB) on May 29,1998 and have been accorded the accession numbers NCIMB 40954 and NCIMB40955, respectively.

In a fiber embodiment of the invention there is provided a probioticcheese ready for consumption which contains a viable, actively growingstrain of L. paracasei as hereinbefore defined in an amount of 10⁷ cfu/gor greater, following manufacture thereof using said L. paracasei as astarter adjunct.

An especially preferred cheese is Cheddar cheese.

Probiotic Cheddar cheeses can be manufactured in accordance with theinvention containing high levels of L. paracasei strains (10⁸ cfu/gcheese) at a relatively low cost to the producer and using identicalmanufacturing procedures. Importantly, we have shown that incorporationof these strains does not impact negatively on cheese quality, includingaroma, flavour and texture. In addition, our results suggest that cheesealso compares very favourably with yoghurt regarding delivery of viablecells to the GIT despite the apparent age difference of the products.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A is a graph of log cfu/g versus time (days) representing survivalof lactobacilli and starter during cheese ripening in Trial 1 asdescribed in Example 2;

FIGS. 1B-1D are RAPD PCR profiles of a representative number ofLactobacillus isolates from each of the cheeses as described in Example2;

FIG. 2A is a graph of log cfu/g versus time (days) representing survivalof lactobacilli and starter during cheese ripening in Trial 2 asdescribed in Example 2;

FIGS. 2B-2E are RAPD PCR profiles of a representative number ofLactobacillus isolates from each of the cheeses as described in Example2;

FIG. 3A is a graph of log cfu/g versus time (days) representing survivalof Lactobacilli and starter during cheese ripening in Trial 3 asdescribed in Example 2;

FIG. 3B depicts RAPD PCR profiles of a representative number ofLactobacillus isolates from Vat 2 cheese as described in Example 2;

FIG. 4A is a graph of log cfu/g versus time (days) representing survivalof Lactobacilli and starter during cheese ripening in Trial 4 asdescribed in Example 2;

FIG. 4B depicts RAPD PCR profiles of a representative number ofLactobacillus isolates from Vat 2 cheese as described in Example 2

FIG. 5 is a urea PAGE of control and experimental Cheddar cheeses aftereight months of ripening as described in Example 5;

FIG. 6A shows the concentration of individual free amino acids inwater-soluble extracts of six month old control and experimental cheesesfound in Trial 1 as described in Example 5; and

FIG. 6B shows the concentration of individual free amino acids inwater-soluble extracts of six month old control and experimental cheesesfound in Trial 2 as described in Example 5.

MODES FOR CARRYING OUT THE INVENTION

The invention will be further illustrated by the following examples:

EXAMPLE 1

Probiotic Strain Identification/enumeration

A pre-requisite to the successful enumeration of added probiotic strainsis to be capable of selectively identifying these from the natural,often complex microflora found in food products. Since NSLAB can reachlevels of up to 10⁷-10⁸ cfu/g in cheese during ripening it was necessaryto evaluate a number of methods aimed at selectively enumerating thelactobacilli added as starter adjuncts from these NSLAB.

The probiotic Lactobacillus strains used in this Example had previouslybeen isolated from the human gastrointestinal tract, and were obtainedfrom Prof. J. K. Collins, Microbiology Dept., University College Cork,Ireland under the aforementioned Materials Transfer Agreement. Thesestrains were identified as L. salivarius (ssp. salivarius) and L.paracasei (ssp. paracasei) by SDS-PAGE analysis of total cell protein.(Reuter, G. (1990) Bifidobacteria microflora 9:107-118) and weredesignated Lb. salivarius NFBC 310, NFBC 321 and NFBC 348 and L.paracasi NFBC 338 and NFBC 364. NSLAB Lactobacillus strains (Lb.curvatus DPC 2042 and 2081, L. plantarum DPC 2102 and 2142 and L. caseissp. casei DPC 2047 and 2103) which had previously been isolated from 8week old commercial Cheddar cheeses, were obtained from the culturecollection of the Dairy Products Research Centre. All Lactobacillusstrains were routinely cultured in MRS broth (Dinakar, P. and V. V.Mistry (1994)) (Difco Laboratories, Detroit, Mich., USA) under anaerobicconditions (anaerobic jars with ‘Anaerocult A’ gas packs; Merck,Darmstadt, Germany) at 30° C. and 37° C. for NSLAB and probioticstrains, respectively. Solid media were prepared by adding 1.5% agar tobroth medium. Stock cultures were maintained at −80° C. in 40%glycerol-supplemented MRS broth. Each culture was sub-cultured twice inMRS broth before use from stock. Lactococcus lactis ssp. cremorisstrains 227 and 223, obtained from Chr. Hansen's Laboratories (LittleIsland, Cork, Ireland) in the form of freeze-dried pellets, were used asstarters for cheese-making. These were grown overnight at 21° C. inheat-treated (90° C. for 30 min) 10% (w/v) reconstituted skim-milk(RSM).

Bacteriological Analyses of Cheeses

Viability of lactobacilli (both probiotic adjuncts and NSLAB) in theinoculated cheese-milk and in the cheeses during ripening was determinedon LBS agar after 5 days of anaerobic incubation at 30° C. while starterlactococci were enumerated on LM17 agar after 3 days incubation at 30°C. Coliforms were enumerated in cheese-milk and cheeses on Violet RedBile Agar (VRBA; Oxoid) at 37° C. for 24 hours. Cheeses were asepticallysampled in duplicate for bacteriological analysis, at intervals duringripening. Cheese samples were emulsified in sterile 2% (w/v) trisodiumcitrate, diluted in maximum recovery diluent and appropriate dilutionspour-plated. After 1, 3 and 6 monthly intervals, 20 individualLactobacillus colonies from each cheese were randomly selected from theLBS agar plates for RAPD-PCR analysis.

a) Bile and Temperature Tolerance of Lactobacillus Adjuncts

To investigate the tolerance of both the probiotic and NSLABLactobacillus isolates to bile, overnight MRS broth cultures of each ofthe Lactobacillus strains were serially diluted in maximum recoverydiluent (Oxoid Ltd, Basingstoke, Hampshire, UK) and appropriatedilutions pour-plated on MRS agar with 0, 0.1, 0.3, 0.5, 1.0 or 3.0%porcine bile (Sigma Chemical Co., Poole, Dorset, England). After 3 daysincubation, the plates were examined and where colonies were present,their numbers and sizes were recorded. Temperature tolerance of theprobiotic lactobacilli was investigated by pour-plating appropriatedilutions of overnight cultures on LBS agar (Rogosa, M. et al. (1951) J.Bateriol 62:132-133) (Becton Dickinson, Cockeysville, Md., USA) andincubating the plates anaerobically both at 37° C. (which is the optimumtemperature of growth for these strains) and at 42° C. Colony numbersobtained after 5 days were compared. In the same way, the temperaturetolerance of these strains and NSLAB, following isolation from Cheddarcheese was also investigated.

The bile and temperature tolerance of both the human-derivedlactobacilli and a selection of NSLAB was first determined in the hopethat either of these parameters could form a basis for the selection ofthe adjunct from the product. Both the probiotic adjuncts and NSLABLactobacillus strains varied considerably with regard to their biletolerance. Two of the NSLAB isolates used in this Example were tolerantto levels of porcine bile of up to 3% compared to the lactobacilli addedas starter adjuncts, which were inhibited at 0.3% bile. Thereforeselections based on bile tolerance would not be useful in distinguishingthe probiotic adjunct lactobacilli incorporated into Cheddar cheese inthis Example from the NSLAB lactobacilli. Similarly, temperaturetolerance could not be used as a basis for selection of the probioticlactobacilli from NSLAB. NSLAB isolated from Irish Cheddar cheeses donot grow at 45° C. while some of the human-derived probioticlactobacilli may withstand such temperatures (Kandler, O. and Weiss, N.(1989) In P. H. A. Sneath (ed.), Bergey's manual of determinativebacteriology, Vol. 2. The Williams & Wilkins Co., Baltimore, Md.). Atemperature of 42° C. was evaluated for selective enumeration of theprobiotic strains from the NSLAB. While the probiotic Lactobacillusstrains, isolated from fresh cultures or Cheddar cheese early inripening were capable of growth at 42° C., they failed to grow at thistemperature when isolated from mature cheese. Furthermore, some NSLABlactobacilli were found to be capable of growth at 42° C., confirmingthat this procedure was non-selective for the human-derived probioticLactobacillus strains from Cheddar cheese.

b) RAPD-PCR Analysis

RAPD-PCR analysis was carried out on each of the probiotic Lactobacillusstrains and on cultures grown from Lactobacillus colonies isolated fromCheddar cheese. Genomic DNA was isolated from 1.5 ml of overnight MRSbroth cultures using a modification of the method of Hoffman and Winston(Hoffman, C. S., and Winston, F. (1997) Gene 57:267-272). This procedureutilises shearing with glass beads to lyse the cells, and was modifiedas outlined by Coakley et al. (Coakley,M. et al. (1996); J. Inst. Brew.102:344-354). One microliter of the extracted DNA was used in subsequentPCR amplifications, which were performed in a total volume of 25 μl in aPerkin-Elmer (Norwalk, Conn., USA) DNA Thermal Cycler. The methodemployed was essentially as described by Coakley et al. ((1996) supra )and used a single primer of arbitrary nucleotide sequence (5′ ATGTAACGCC3′), obtained from Pharmacia Biotech, (Uppsala, Sweden). DNA wasamplified for 35 cycles using the following temperature profile:denaturing at 93° C. for 1 min, annealing at 36° C. for 1 min followedby polymerisation at 72° C. for 1 min. Taq DNA polymerase (0.625 Units,Bioline) was added to the reaction mix during the first denaturationstep (Hot Start).

Between 5 and 10 μl of the PCR reaction was analysed on a 1.5% (w/v)agarose (Sigma) gel with ethidium bromide staining. A 100 bp ladder(Pharmacia) was used as a molecular weight standard. Gels were run forapproximately 3 hours at 100 V and the DNA visualised by UVtransillumination.

Consequently, the Randomly Amplified Polymorphic DNA (RAPD) method,which involves PCR using an arbitrary primer, was used to generate DNAfingerprints for each of the probiotic strains. Each of theLactobacillus strains generated reproducible discrete DNA fingerprints,which were found to be substantially different from those ofrepresentative NSLAB lactobacilli. Thus, the RAPD method proved to be asuccessful means of identifying the probiotic strains and demonstratedpotential as a means of selective identification of the strains from theNSLAB flora in Cheddar cheese.

EXAMPLE 2

Incorporation of Lactobacillus Species into Cheddar Cheese

Laboratory-scale cheesemaking trials (Trials 1 and 2) were performedinitially using 25 L of pasteurised whole milk in each cheese vat. Tolimit contamination with wild lactobacilli, these cheeses weremanufactured under controlled bacteriological conditions, as describedby McSweeney, P. et al. ((1994); Irish J. Agric. Food Res. 33:183-192).A 1.5% inoculum of the mixed-strain starter culture was used and in eachtrial one vat (Vat 1) acted as a control to which starter only wasadded. To each of the experimental vats, one probiotic Lactobacillusstrain, grown overnight in 10% RSM, was added as an adjunct to thestarter culture. In Trial 1, the probiotic adjuncts L. salivarius NFBC348 and L. paracasei NFBC 364 were added at an inoculum level of 0.1% toVats 2 and 3, respectively. In the second trial, L. salivarius NFBC 310(Vat 2), L. salivarius NFBC 321 (Vat 3) and L. paracasei NFBC 338 (Vat4) were inoculated at a level of 0.2%. Cheddar cheeses were thenmanufactured according to standard procedures as follows:Filter-sterilised rennet (Chr. Hansen's Laboratories) was added at aconcentration of 0.07 ml/liter 35 min after starter and adjunctaddition, and the curd was cut approximately 40 min later. Curds werecooked to 39° C., pitched at pH 6.1 and milled at approximately pH 5.3.Salt was added at a rate of 2.8% (w/w) and the curds were placed inmoulds and pressed at approximately 200 kPa overnight. The cheeses wereremoved from the moulds, vacuum-packed and ripened at 8° C. forapproximately 8.5 months. Subsequently two pilot-scale cheesemakingtrials (Trials 3 and 4) were performed using two of the adjunctLactobacillus strains which were found to maintain high viability in thelaboratory-scale cheeses during ripening. In each trial, two vats, oneexperimental and one control, each containing 450 liters of standardised(fat:protein=1) pasteurised whole milk were used. As in thelaboratory-scale trials, a 1.5% inoculum of the starters 223 and 227 wasadded to each vat. In addition, in each trial the experimental vat (Vat2) contained a 0.1% inoculum of either L. paracasei NFBC 364 (Trial 3)or NFBC 338 (Trial 4) added as a starter adjunct. The cheesemakingprocedure was as previously described for the laboratory-scale cheesesexcept that the salting level was 2.7% and the curds were pressedovernight at approximately 413 kPa.

Initially, laboratory-scale cheese trials were performed undermicrobiologically controlled conditions (thus limiting development ofhigh numbers of NSLAB during ripening) to assess the performance of fiveprobiotic Lactobacillus strains in Cheddar cheese. Firstly, forinoculation purposes, the performance of these strains in RSM wasinvestigated. None of the strains performed well in milk (levels of only10⁷-10⁸ cfu/ml achieved) and were subsequently found to be non- or onlyweakly proteolytic (data not shown). Thus, using a 0.1-0.2% inoculum ofthese L. salivarius and paracasei strains as starter adjuncts,relatively low levels of 10⁴-10⁵ cfu/ml milk were obtained in theexperimental vats during cheese manufacture as shown in Table 1. Alladjunct lactobacilli were found to survive the cheese manufacturingprocess and, given their poor growth in milk and the low inoculum used,were shown to have no effect on acid production during the process (datanot shown). Results demonstrate that cheese made with NFBC 364 and NFBC338 L. paracasei adjuncts (Trial 1 Vat 3, Trial 2, Vat 4, respectively)contained high levels of these probiotic strains after 8 months ofripening; with final counts of 9.2×10⁷ and 1.4×10⁸ cfu/g achieved,respectively as shown in FIGS. 1A and 2A.

TABLE 1 Baterial counts (cfu/ml) in milk used for the manufacture ofCheddar cheese, after inoculation with adjunct and/or starter culturesCheese inoculum¹ Lactobacilli Lactococci Trial 1³ V1, 1.5% 227/223 ND²3.2 × 10⁶ V2, 1.5% 227/223 + 0.1% L. salivarius 1.3 × 10⁵   3 × 10⁶ NFBC348 V3, 1.5% 227/223 + 0.1% L. paracasei 2.4 × 10⁵ 2.9 × 10⁶ NFBC 364Trial 2³ V1, 1.5% 227/223 ND 2.7 × 10⁶ V2, 1.5% 227/223 + 0.2% L.salivarius 2.9 × 10⁵ 3.8 × 10⁶ NFBC 310 V3, 1.5% 227/223 + 0.2% b.salivarius   2 × 10⁵ 2.8 × 10⁶ NFBC 321 V4, 1.5% 227/223 + 0.2% L.paracasei 2.3 × 10⁴ 4.5 × 10⁵ NFBC 338 Trial 3⁴ V1, 1.5% 227/223 ND 2.4× 10⁶ V2, 1.5% 227/223 + 0.1% L. paracasei 1.7 × 10⁵ 4.1 × 10⁶ NFBC 338Trial 4⁴ V1, 1.5% 227/223 ND 3.10⁵ V2, 1.5% 227/223 + 0.1% L. paracasei8.9 × 10⁵ 1.1 × 10⁶ NFBC 364 ¹227/223 = L. Lactis ssp. cremoris 227 +223 ²ND = Non-detectable ³Trial 1 and 2 cheeses manufactured atlaboratory-scale under microbiologically controlled conditions ⁴Trial 3and 4 cheeses manufactured at pilot-scale using the two Lactobacillusadjunct strains (NFBC 338 and NFBC 364) which showed good survival inthe laboratory-scale cheeses during ripening

FIGS. 1B-1D are RAPD PCR profiles of a representative number ofLactobacillus isolates from each of the cheeses (B, C and D); Lane 1shows the RAPD profile of the probiotic Lactobacillus strain added tothe cheese at manufacture, while a 100 bp ladder is shown at Lane 19 (B)and Lane 11 (C and D) and all other lanes (B, C and D) show RAPDprofiles of Lactobacillus isolates from 6-month-ripened cheeses.

FIGS. 2B-2D are RAPD PCR profiles of a representative number ofLactobacillus isolates from each of the cheeses (B, C, D and E); Lane 1shows the RAPD profile of the probiotic Lactobacillus strain added tothe cheese at manufacture, while a 100 bp ladder is shown in Lane 19 (B,C, D and E) and all other lanes (B, C, D, and E) show RAPD profiles ofLactobacillus isolates from 6-month-ripened cheeses.

The high levels of probiotic strains was confirmed following comparisonof the RAPD PCR fingerprints generated for L. paracasei strains NFBC 364and NFBC 338 (FIG. 1D and FIG. 2E, lane 1) and those obtained forlactobacilli isolated from the cheeses (FIG. 1D and FIG. 2E, lanes 2-10and 12-20) which were found to be identical. In contrast, althoughlactobacilli grew to high levels (1×10⁸ cfu/g) in the cheese to whichstrain NFBC 310 was added (Trial 2 Vat 2), and subsequently remained atthis level throughout ripening (FIG. 2A), these lactobacilli (FIG. 2C,lanes 2-10 and 12-2-) were identified by RAPD PCR as NSLAB. Levels oflactobacilli in cheeses with L. salivarius adjuncts NFBC 348 and NFBC321 (Trial 1 Vat 2 and Trial 2 Vat 3, respectively) declined to 1.2×10⁵cfu/g and 8.6×10⁴, respectively, after 4 months of ripening (FIGS. 1Aand 2A), although these levels did increase slightly to reach finallevels of 3.5×10⁵ and 1.1×10⁶ cfu/g, respectively after 8 months ofripening. Interestingly, the genetic fingerprints of isolates taken fromeach of these cheeses after 6 months revealed that these lactobacilliwere predominantly NSLAB (FIG. 1C and FIG. 2D, respectively). Thus, theL. salivarius strains used in this Example did not maintain viability inCheddar cheeses during ripening. Furthermore, many of the NSLAB isolatedfrom these cheeses in which the adjunct strains declined (FIG. 1C, lanes3-6 and FIG. 3D, lanes 12-18) and from the control cheeses to which noprobiotic adjuncts were added (FIG. 1B, lanes 9-13 and FIG. 2B, lanes3-9) yielded identical PCR-generated DNA fingerprints. This suggeststhat the DNA was obtained from identical strains and shows apredominance of certain Lactobacillus strains in the NSLAB population ofthese cheeses.

Subsequently, pilot-scale cheese trials were performed, where only thetwo L. paracasei strains, NFBC 338 and NFBC 364, which survived to highlevels in the laboratory-scale trials were incorporated into Cheddarcheese. These strains were added to Trial 3 Vat 2 (NFBC 338) and Trial 4Vat 2 (NFBC 364) at inocula of 1.7 and 8.9×10⁵ cfu/ml cheese-milk,respectively as shown in Table, 1. Thereafter, both NFBC 338 and NFBC364 grew in the cheese from initial numbers of 1.1.×10⁷ and 2.7×10⁷cfu/g, respectively, to reach levels of between 1.5 and 2.9×10⁸ cfu/gafter 3 months of ripening and viability was sustained at this level forthe remainder of the ripening period (FIGS. 3A and 4A). As in thelaboratory-scale cheeses, these results were confirmed by RAPD PCRanalysis (as described in Example 1) of a number of isolates from eachof these cheeses (FIGS. 3B and 4B).

Taken together, the data from the laboratory- and pilot-scale cheesetrials provide molecular-based evidence for the persistence in Cheddarcheese of strains selected for their potential as probiotics. In orderto appreciate the beneficial effects of ‘probiotic’ foods, it has beenproposed as indicated above, that viable probictic organisms should bepresent at levels of at least 10⁷ viable cells per gram or milliliter ofproduct. The probiotic-containing cheeses obtained in accordance withthe invention contained levels of up to 10⁸ cfu/g cheese, thussatisfying the criteria for a ‘probiotic’ food product.

It should also be noted that lactococcal starter numbers in the controlcheeses of all trials showed a typical decline during the ripeningperiod (FIGS. 1A, 2A, 3A and 4A). However, due to the growth oflactobacilli on the LM17 medium used to enumerate, these starterorganisms, it was possible only to monitor starter in these cheeses, towhich no adjunct lactobacilli had been added, and then only in the earlystages of ripening.

RAPD PCR analysis, when used as an identification method, was capable ofdetermining that probiotic L. paracasei strains grew and maintained highviability (10⁸ cfu/g) in cheese, while the particular L. salivariusadjunct strains used did not appear to be suited for such anapplication. Furthermore, survival of these probiotic Lactobacillusstrains at high numbers in Cheddar cheese was achieved using arelatively low inoculum (0.1-0.2%) in the cheese vat and withoutaltering the cheesemaking process in any way. This was possible becausethese strains were added as starter adjuncts and were not thereforenecessary for acid production during cheese-making. Thus, the processaccording to the invention for incorporation of probiotic organisms intoCheddar cheese offers certain advantages to industry; no alteration ofexisting cheese-making technology and low cost due to the low inoculumrequired.

EXAMPLE 3

Cheese Compositional Analysis

Grated cheese samples were analysed in duplicate for salt by apotentiometric method (Irish Dairy Federation (1979); Cheese andprocessed cheese. Determination of chloride content: potentiometrictitration method. IDF Standard 88), fat by the Gerber method (IrishStandard (1955); Determination of the percentage fat in cheese. IrishStandard. 69), moisture by oven-drying at 102° C. (Irish DairyFederation (1982); Determination of the total solids content (cheese andprocessed cheese). IDF Standard 4A) and protein on a LECO FP-428nitrogen determinator. The pH of a slurry, prepared by blending 12 mlH₂O with 20 g grated cheese, was measured using a standard pH meter(Radiometer, Copenhagen, Denmark).

The composition of the cheese was generally found to be within the rangetypical for Cheddar as shown in Table 2.

TABLE 2 Composition¹ of control and probiotic Cheddar cheeses MoistureSalt S/M² Fat Protein pH Cheese trial (%) Trial 1 V1 38.28 1.53 4.0 31.526.33 5.4 V2 38.24 1.70 4.45 32.0 26.63 5.2 V3 39.89 1.23 3.08 31.025.79 5.3 Trial 2 V1 37.48 1.64 4.38 33.0 26.5 5.2 V2 35.73 1.81 5.0733.0 26.99 5.1 V3 37.22 1.61 4.33 33.0 27.27 5.1 V4 38.01 1.71 4.55 33.027.27 5.1 Trial 3 V1 35.61 1.76 4.94 33 26.33 5.2 V2 36.74 1.72 4.68 3326.56 5.2 Trial 4 V1 34.88 2.05 5.88 34.5 26.17 5.4 V2 35.14 1.80 5.1235.0 26.42 5.3 ¹Means of duplicate analyses ²Salt-in-moisture

Some atypical values for salt-in-moisture (Vat 3), fat (all vats) and pH(Vat 1) were obtained for the Trial 1 cheeses which reflects thedifficulty in controlling the cheesemaking parameters (i.e. temperature)at a laboratory-scale. In contrast, all the compositional analysisvalues obtained for the pilot-scale trials were generally within thetypical range for Cheddar. Thus, the comparable values observed forcontrol and experimental cheeses (Table 2) indicate that incorporationof probiotic lactobacilli as starter adjuncts, and their survival athigh numbers, had no direct effect on cheese composition.

EXAMPLE 4

Sensory Evaluation of Cheddar Cheese

Cheeses were graded blindly after 3 and 6 months ripening by acommercial grader from a local cheese manufacturing plant. The cheeseswere graded for flavour/aroma and body/texture, with maximum scores of45 and 40, respectively. Minimum scores of 38 and 31 for flavour/aromaand body/texture, respectively are required for commercial Cheddarcheese. With the exception of the control cheese of Trial 2, all cheesescould be described as commercial grade with respect to sensory criteria,after 6 months of ripening, having achieved minimum scores of 38 and 31for flavour/aroma and body/texture, respectively as shown in Table 3.

TABLE 3 Sensory evaluation of Cheddar cheeses at 6 months CheeseFlavour/aroma¹ Body/texture² Trial 1 V1 38 33 V2 38 33 V3 39 32 Trial 2V1 37 33 V2 39 32 V3 38 33 V4 38 32 Trial 3 V1 38 33 V2 38 33 Trial 4 V139 33 V2 38 33 ¹Maximum score = 45; minimum commerical score = 38²Maximum score = 40; minimum commercial score = 31Lactobacillus adjuncts have previously been reported to improve Cheddarcheese flavour (Broome, M. D., et al. (1990); Aust. J. Dairy Technol.45:67-73) although, in some cases they were responsible for flavourdefects (Puchades, R., et al. (1989); J. Food Sci. 54:885-888). In thisExample, laboratory-scale cheeses with high levels of Lactobacillusadjuncts were found to have flavour and texture comparable to that ofcontrol cheeses, indicating that addition of these probioticlactobacilli to Cheddar cheese had no adverse effects on sensorycriteria. Furthermore, when repeated on a larger scale, sensoryparameters remained unaffected by the presence of high levels of theseadjuncts.

EXAMPLE 5

Proteolysis in Laboratory-scale Cheddar Cheeses

Cheeses were analysed by urea-PAGE (Shalabi, S. L., and Fox, P. F.(1987); Irish J. Food Sci. Technol. 11:135-151) using a Protean II xivertical slab gel unit (Bio-Rad Laboratories, Ltd, Watford, Herts, UK)essentially with the stacking gel system of Andrews (Andrews, A. T.(1983); J. Dairy Res. 50:45-55). Cheese samples were prepared bydispersing 10 mg of grated cheese in 1 ml of sample buffer and heatingat 50° C. for 5 min. Samples were stored at −20° C. until use and 10 μlwas applied to the gel. Sodium caseinate (5 μl ) was used as a standardfor comparative purposes. Samples were electrophoresed at 280 V throughthe stacking gel and at 300 V through the resolving gel. Gels werestained with Coomassie Brilliant Blue G250 using the direct-stainingprocedure of Blakesley and Boezi (Blakesley, R. W., and J. A. Boezi(1977); Anal. Biochem 82:582-581).

Water-soluble extracts (pH 4.6) of each of the cheeses were preparedaccording to the method of Kuchroo and Fox (Kuchroo, C. N. and Fox, P.F.; (1982); Milchwissenshcaft 37:331-335) and freeze-dried. The sizedistribution of peptides in these freeze-dried extracts was determinedby size-exclusion HPLC, using a TSK 2000 SW (Beckman Instruments Ltd,High Wickham, United Kingdom) gel permeation column (7.5 nm×60 cm)fitted to a Waters HPLC system (Waters Chromatography Division, Milford,Mass., USA). The column was eluted at a flow-rate of 1 ml/min with 30%acetonitrile containing 0.1% trifluoroacetic acid (TFA). Thefreeze-dried water-soluble extracts were reconstituted (3 mg/ml) inHPLC-grade water, filtered through a Whatman 0.2 μm filter and 20 μlapplied to the column. Column eluates were continually monitored at 214nm. Data were collected using a PC Minichrom system (VG Data Systems,Cheshire, United Kingdom) and results compared to a previously preparedcalibration curve.

Individual free amino acids (FAA) in the water-soluble extracts weredetermined using a Beckman System 6300 High Performance Analyser(Beckman Instruments Ltd, High Wickham, United Kingdom) equipped with aBeckman P-N 338052 Na⁺ column (12 cm×0.5 cm) as described by Lynch etal.(Lynch, C. M. et al. (1996); Int. Dairy J. 6:851-867). Chromatogramswere collected using a computer-controlled Minichrom data processingpackage. Amino acid concentrations were expressed as μg/ml cheeseextract which were subsequently converted to μg/gcheese.

Urea-PAGE electrophoresis patterns of whole cheese samples after 8months of ripening (FIG. 5) are typical for Cheddar and do not show anydifferences in the extent of primary proteolysis between the controlcheeses and those manufactured with adjunct lactobacilli.

FIG. 5 represents Urea-PAGE of control (Lanes 2 and 5) and experimental(Lanes 3, 4, 6, 7 and 8) Cheddar cheeses after 8 months of ripening.Lane 1 contains a sodium-caseinate standard.

The molecular weight distribution of peptides in water-soluble extractsfrom the cheeses (as measured by size-exclusion HPLC) serves as afurther indication of the extent of proteolysis in the cheeses duringripening; the greater the extent of proteolysis, the higher the level oflow molecular weight peptides generated. After 6 months of ripening, thelevels of these low molecular weight peptides (<500 Da) were found tohave accumulated to high levels in all cheeses (data not shown).Moreover, similar levels were detected in the control and experimentalcheeses, even in those cheeses which had high levels of survival ofadjunct lactobacilli (Trial 1 Vat 3, Trial, 2 Vat 4 cheeses), indicatingthat the extent of proteolysis in the cheeses as demonstrated bygeneration of small peptides, was not affected by adjunct addition.However higher levels of individual FAA were detected in the cheesesmade with added lactobacilli, after 6 months of ripening (FIG. 6).

FIG. 6 depicts the concentration of individual free amino acids inwater-soluble extracts of 6 month old control and experimental Cheddarcheeses of Trial 1 (A) and Trial 2 (B).

Most notably, concentrations of serine, methionine, leucine andphenlyalanine (Trial 1) in addition to glutamnic acid and valine (Trial2) were higher in the cheeses made with added lactobacilli than in thecontrol cheese to which no adjunct had been added (FIG. 6). This wasfound to be true even for the cheeses in which the Lactobacillusadjuncts declined during ripening. This may be accounted for by therelease of intracellular peptidases as the organisms died and lysed.Thus, in general, the results suggest that the adjunct lactobacilli,whether they survived to high levels or not, did contribute toproteolysis in the cheese as demonstrated by increased formation of FAA.

The above results demonstrate that probiotic L. paracasei strains,incorporated into Cheddar cheese proved particularly suitable as starteradjuncts. These strains were found to grow and proliferate to high cellnumbers in the cheese over 8 months of ripening, even when added at arelatively low inoculum. Furthermore, RAPD PCR proved extremely usefulto distinguish these probiotic adjuncts from NSLAB. Moreover, theresults from the control cheese suggest the predominance of certainNSLAB strains. While proteolysis during cheese ripening was influencedby the adjuncts at the level of FAA formation, cheese flavour, textureand appearance were not affected. Incorporation of these probioticadjuncts into Cheddar cheese, as described herein can be achievedwithout alteration of the cheesemaking technology, thus making thissystem attractive for commercial exploitation. These results indicatethat Cheddar cheese is an effective vehicle for delivery of thesestrains to the consumer with the attendant advantages.

1. A process for the manufacture of a probiotic cheese, which processcomprises adding a 0.05-0.5% vol/vol inoculum of a strain ofLactobacillus paracasei, which is non-pathogenic, acid and bile tolerantand adherent to human epithelial cells, as a starter adjunct to cheesemilk, said L. paracasei strain being capable of growing during theripening phase to a level of 10⁷ cfu/g or greater.
 2. A processaccording to claim 1, wherein a 0.1-0.25% vol/vol inoculum of the L.paracasei is added to the cheese milk.
 3. A process according to claim 1or 2, wherein the ripening phase is at least six months.
 4. A processaccording to claim 1, wherein the ripening phase is eight months orgreater.
 5. A process according to claim 1, wherein the L. paracasei iscapable of growing during the ripening phase to a level of 10⁸ cfu/g orgreater.
 6. A process according to claim 1, wherein the L. paracasei istolerant to temperatures of 37° C. or greater.
 7. A process according toclaim 1, wherein the L. paracasei can be enumerated and distinguishedfrom the resident flora.
 8. A process according to claim 7, wherein theadded L. paracasei cells are enumerated and distinguished by a randomlyamplified polymorphic DNA (RAPD) method which allows the generation ofdiscrete DNA fingerprints for the respective strains.
 9. A processaccording to claim 1, wherein the cheese manufactured is a hard cheese.10. A process according to claim 9, wherein the cheese is Cheddarcheese.
 11. Lactobacillus paracasei strain NFBC
 338. 12. Lactobacillusparacasei strain NFBC
 364. 13. A probiotic cheese ready for consumptionwhich contains a viable, actively growing strain of L. paracasei asdefined in claim 1 in an amount of 10⁷ cfu/g or greater, followingmanufacture thereof using said L. paracasei as a starter adjunct.
 14. Aprobiotic cheese according to claim 13, which is Cheddar cheese.